Project Summary The thymus is an organ essential for the development of T cells. There are several congenic disorders in which the thymus fails to develop, resulting in a severe combined immunodeficiency in newborns. These include infants with autosomal recessive mutations in FOXN1 or PAX1, and a subset of newborns with chromosome 22q11.2 deletion syndrome (DiGeorge). For any born with a complete aplasia of the thymus due to these mutations, an allogeneic thymic stromal tissue transplant is the clinical approach for restoring some T cell development. Patients with mutations in FOXN1 present with an athymia along with a now classically described nude phenotype due to a failure of the hair follicle to penetrant through the dermal layer. As FOXN1 is the master transcription factor responsible for thymic epithelial cell development, these patients have no T cells. They are T-B+NK+ SCID/Nude. With the advent of targeted exome and whole genome sequencing, we report on multiple infants born with compound heterozygous FOXN1 mutations. This results in a new phenotype of T-B+NK+ with normal hair growth and nail bed extrusion. Since it is impossible to determine causal effects of the diverse compound heterozygous mutations in the human cohort, we are genocopying the mutations in mouse models using CRISPR/Cas9. Several mouse lines studied to date have phenocopied the human T-B+NK+ SCID with normal hair follicle development. With 15 additional and distinct mutations in human FOXN1 identified, we will define the functional impact of these mutations on the protein. This will include transcriptional reporter assays, structural studies, and analyses of cortical and medullary thymic epithelial development. Our ultimate goal is to understand the differential roles of FOXN1 in thymic versus keratinocyte development. This will enable clinicians to understand genotype/phenotype relationships of FOXN1 structure and function to determine when an affected infant requires an allogeneic thymic tissue transplant. !